A chloride-translocating adenosine triphosphatase in Acetabularia acetabulum. 1. Purification and characterization of a novel type of adenosine triphosphatase that differs from chloroplast F1 adenosine triphosphatase

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Abstract
ATPases were solubilized from membranes of Acetabularia acetabulum using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. Three fractions of ATPase, Mono Q-I, -II, and -III, were separated. Activity in fraction Mono Q-I was very labile and could not be accurately determined. Fractions Mono Q-II and -III had specific activities of 0.6 and 6 units/mg of protein, respectively. By SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping, it was shown that fractions Mono Q-II and -III consisted of the same polypeptides with molecular masses of 54K (a-subunit) and 50K (b-subunit). Fractions Mono Q-II and -III had the following catalytic properties: pH optimum at 6.0; substrate specificity, ATP = GTP = ITP .mchgt. UTP = CTP (Km for ATP 0.6 mM); divalent cation requirement, Mn2+ = Mg2+ > Co2+ > Zn2+ .mchgt. Ca2+, Ni2+. Both activities were inhibited by monovalent anions, while monovalent cations had neither inhibitory nor stimulatory effects. Orthovanadate inhibited both activities to 50% at 1 mM, and the most effective inhibitor of both was azide (95% inhibition at 100 .mu.M). An enzyme-phosphate complex was formed after incubation of fraction Mono Q-III with [.gamma.-32P]ATP. The CF1-ATPase subcomplexes were isolated from the same organism and compared with the fraction Mono Q-III. Data supported the difference of fraction Mono Q-III from CF1-ATPase.